It is well known that most of the bodily states in multicellular organisms, including most disease states, are effected by proteins. Such proteins, either acting directly or through their enzymatic or other functions, contribute in major proportion to many diseases and regulatory functions in animals and man. For disease states, classical therapeutics have generally focused upon interactions with such proteins in efforts to moderate their disease-causing or disease potentiating functions. In newer therapeutic approaches, modulation of the actual production of such proteins is desired. By interfering with the production of proteins, the maximum therapeutic effect may be obtained with minimal side effects. It is therefore a general object of such therapeutic approaches to interfere with or otherwise modulate gene expression, which would lead to undesired protein formation.
One method for inhibiting specific gene expression is with the use of oligonucleotides, especially oligonucleotides that are complementary to a specific target messenger RNA (mRNA) sequence. Generally, nucleic acid sequences complementary to the products of gene transcription (e.g., mRNA) are designated “antisense”, and nucleic acid sequences having the same sequence as the transcript or being produced as the transcript are designated “sense”. See, e.g., Crooke, 1992, Annu. Rev. Pharmacol. Toxicol., 32: 329-376. An antisense oligonucleotide can be selected to hybridize to all or part of a gene, in such a way as to modulate expression of the gene. Transcription factors interact with double-stranded DNA during regulation of transcription. Oligonucleotides can serve as competitive inhibitors of transcription factors to modulate their action. Several recent reports describe such interactions (see Bielinska, A., et al., 1990, Science, 250: 997-1000; and Wu, H., et al., 1990, Gene 89: 203-209).
Molecular strategies are being developed to down-regulate unwanted gene expression. Recently, the use of modified oligonucleotide compounds has developed into a promising method of treatment against such diseases as viral infections, inflammatory and genetic disorder and significantly, cancer. Antisense DNAs were first conceived as alkylating complementary oligodeoxynucleotides directed against naturally occurring nucleic acids (Belikova, et al., Tetrahedron Lett. 37:3557-3562, 1967). Zamecnik and Stephenson were the first to propose the use of synthetic antisense oligonucleotides for therapeutic purposes. (Zamecnik & Stephenson, 1978, Proc. Natl. Acad. Sci. U.S.A., 75:285-289; Zamecnik & Stephenson, 1978, Proc. Natl. Acad. Sci. U.S.A., 75:280-284). They reported that the use of an oligonucleotide 13-mer complementary to the RNA of Rous sarcoma virus inhibited the growth of the virus in cell culture. Since then, numerous other studies have been published manifesting the in vitro efficacy of antisense oligonucleotide inhibition of viral growth, e.g., vesicular stomatitis viruses (Leonetti et al., 1988, Gene, 72:323), herpes simplex viruses (Smith et al., 1987, Proc. Natl. Acad. Sci. U.S.A. 83:2787), and influenza virus (Seroa; et al., 1987, Nucleic Acids Res. 15:9909).
Oligonucleotides have also found use in among others, diagnostic tests, research reagents e.g. primers in PCR technology and other laboratory procedures. Oligonucleotides can be custom synthesized to contain properties that are tailored to fit a desired use. Thus numerous chemical modifications have been introduced into oligomeric compounds to increase their usefulness in diagnostics, as research reagents and as therapeutic entities.
Although oligonucleotides, especially antisense oligonucleotides show promise as therapeutic agents, they are very susceptible to nucleases and can be rapidly degraded before and after they enter the target cells making unmodified antisense oligonucleotides unsuitable for use in in vivo systems. Because the enzymes responsible for the degradation are found in most tissues, modifications to the oligonucleotides have been made in an attempt to stabilize the compounds and remedy this problem. The most widely tested modifications have been made to the back-bone portion of the oligonucleotide compounds. See generally Uhlmann and Peymann, 1990, Chemical Reviews 90, at pages 545-561 and references cited therein. Among the many different back bones made, only phosphorothioate showed significant antisense activity. See for example, Padmapriya and Agrawal, 1993, Bioorg. & Med. Chem. Lett. 3, 761. While the introduction of sulfur atoms to the back bone slows the enzyme degradation rate, it also increases toxicity at the same time. Another disadvantage of adding sulfur atoms is that it changes the back bone from achiral to chiral and results in 2n diastereomers. This may cause further side effects. Still more disadvantages of present antisense oligonucleotides are that they may carry a negative charge on the phosphate group which inhibits its ability to pass through the mainly lipophilic cell membrane. The longer the compound remains outside the cell, the more degraded it becomes resulting in less active compound arriving at the target. A further disadvantage of present antisense compounds is that oligonucleotides tend to form secondary and high-order solution structures. Once these structures are formed, they become targets of various enzymes, proteins, RNA, and DNA for binding. This results in nonspecific side effects and reduced amounts of active compound binding to mRNA. Other attempts to improve oligonucleotide therapy have included adding a linking moiety and polyethylene glycol. See for example, Kawaguchi, et al., Stability, Specific Binding Activity, and Plasma Concentration in Mice of an Oligodeoxynucleotide Modified at 5′-Terminal with Poly(ethylene glycol), Biol. Pharm. Bull., 18(3) 474-476 (1995), and U.S. Pat. No. 4,904,582. In both of these examples, the modifications involve the use of linking moieties that are permanent in nature in an effort to stabilize the oligonucleotide against degradation and increase cell permeability. However, both of these efforts fail to provide any efficacy.
Due to the inadequacies of the present methods, there exists a need to improve stability and resistance to nuclease degradation as well as decrease toxicity and increase binding affinity to mRNA of oligonucleotide compounds. The current oligonucleotide therapy is expensive. This is mainly due to the degradation problem. Thus, there is a real need to protect the antisense oligonucleotide compounds against degradation, prevent the formation of high-order structures and at the same time deliver sufficient amounts of active antisense oligonucleotide compounds to the target. This invention provides such improvements.